The use of nanoparticles in in vivo or in vitro diagnostics, in therapy, in embryology and generally in many molecular biological or biochemical applications and also in drug screening has gained more and more importance in recent years. To this end, most applications require nanoparticles which have a hydrophilic surface in order to disperse them in biological environs, i.e. in an aqueous environment. Said nanoparticles should ideally be in a monoparticulate or monodispersed, i.e. non-agglomerated, form, in order to firstly prevent unwanted sedimentation and/or secondly influence as little as possible the dynamics or kinetics of biochemical or biomolecular processes or motions. Furthermore, said nanoparticles should ideally have functional, reactive chemical groups enabling functional molecules to be coupled thereto. Functional molecules may be, for example, biological macromolecules such as, for example, oligonucleotides (e.g. DNA or RNA) or polypeptides (e.g. proteins or antibodies), biological coupling molecules such as, for example, biotin or streptavidin, or other organic molecules.
Fluorescent, inorganic nanoparticles are frequently prepared in organic solvents, resulting in hydrophobic final products. The most commonly used fluorescent, inorganic nanoparticles are semiconductor nanoparticles consisting of II-VI or semiconductors which usually have a core-shell structure. U.S. Pat. No. 6,322,901, U.S. Pat. No. 6,576,291 and U.S. Pat. No. 6,423,551 describe these particles whose inorganic core has a size of less than 10 nm and which are also referred to as “quantum dots”. Due to their preparation, they frequently have an organic shell consisting of trioctylphosphine.
Another class of fluorescent, inorganic nanoparticles are phosphorescent nanoparticles which consist of nonconductive materials and which are doped with ions of the rare earth and/or transition group elements.
They are also referred to as nanophosphors, with WO 04/046035 A1, WO 02/020695 A1, K. Koempe; H. Borchert; J. Storz; A. Arun; S. Adam; T. Moeller; M. Haase; Angewandte Chemie, International Edition (2003), 42(44), 5513-5516 describing “down-converting nanophosphors” whose emission wavelength is longer than that of excitation, and in S. Heer; O. Lehmann; M. Haase; H. Guedel; Angewandte Chemie, International Edition (2003), 42(27), 3179-3182 describing “up-converting phosphors” whose emission wavelength is shorter than that of excitation.
WO 01/86299 A1, WO 03/040024 A1 describe the use of such nanophosphors also as biolabels.
WO 02/020695 A1 describes nanophosphors made of CePO4:Tb and their preparation, for example in tris-ethyl hexyl phosphate (TEHP), producing particles comprising TEHP adhering to their surface. It is also possible to use tributyl phosphate or other hydrophobic derivatives of the phosphates rather than TEHP. While nanoparticles produced in this way cannot be dispersed in water, they can be dispersed in organic solvents, i.e. converted to monoparticulate suspensions.
However, a hydrophilic surface of the nanophosphors is an absolute requirement for the intended application in biological systems. The hydrophilization of nanoparticles having a hydrophobic surface is known in principle, as described in WO 02/055186 (Quantum Dot Corp.). There, hydrophilization of the hydrophobic nanoparticles is carried out with the aid of amphiphilic dispersers which are prepared, for example, by partially reacting polyacrylic acid with octylamine.
In the aqueous phase, the hydrophobic octylamide side chains interact with the hydrophobic surface of the nanoparticles, while the free acrylic acid groups of the amphiphilic disperser are oriented toward the aqueous phase. To the acrylic acid residues oriented in this manner, further molecules, for example, proteins or other biological macromolecules, can be attached via covalent bonds. The amphiphilic disperser here serves as a linker.
A disadvantage of this method is the relatively complex preparation of the hydrophobicized polyacrylic acid derivatives, which are utilized as amphiphilic dispersers, in a reproducible quality and also the relatively large space needed, due to the hydrophobic interaction of the hydrophobic surface of the nanoparticles with the octylamide groups of the amphiphilic polymeric disperser. Nanoparticles modified in this way have a greatly increased average particle size in comparison with the unmodified primary particles, even in the case of monoparticulate dispersion. This increase in volume is disadvantageous for various biological applications in which, for example, the labeled molecules are intended to penetrate biomembranes (e.g. cell wall) or diffuse through channel proteins. It is particularly disadvantageous for the use in homogeneous assays in which a (fluorescence) resonance energy transfer, (F)RET, to a (F)RET partner in spatial proximity is involved in the optical evaluation.
Based on the abovementioned prior art, it is therefore the object to provide inorganic nanoparticles having a hydrophilic surface, without a large increase in the average particle diameter, which nanoparticles preferably have reactive, functional chemical groups, thereby allowing functional molecules to be coupled thereto, and which can be used for biological, molecular biological, biochemical and medical, such as, for example, for diagnostic and therapeutic applications, in particular in homogeneous biological assays based on resonance energy transfer processes, and which, at the same time, are inexpensive and can be readily prepared.